APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1984, p. 936-943 Vol. 48, No. 50099-2240/84/110936-08$02.00/0Copyright © 1984, American Society for Microbiology

Isolation and Characterization of a New Cytophaga SpeciesImplicated in a Work-Related Lung Disease

CYNTHIA A. LIEBERT,1 MARY A. HOOD,'* FRED H. DECK,2 KAREN BISHOP,2 AND DENNIS K. FLAHERTY3Department of Biology, University of West Florida, Pensacola, Florida 325141; Analytical Services, Monsanto Fibers and

Intermediates Co., Pensacola, Florida 325752; and Monsanto Environmental Health Laboratory, St. Louis, Missouri63110'

Received 12 December 1983/Accepted 7 August 1984

A yellow-pigmented, gram-negative, gliding bacterium isolated from an industrial water spray airhumidification system was implicated as a causative agent in several occurrences of lung disease withhypersensitivity pneumonitis-like symptoms. The bacterium, designated WF-164, lacked microcysts or fruitingbodies and had a DNA base composition of 34.8 mol% of guanine plus cytosine. Gliding, flexing, nonflagellatedcells measuring 0.3 by 3.5 to 8.9 ,um were observed by using light and electron microscopy. Tests to determineutilization of selected carbohydrates revealed an amylolitic, chitinoclastic, noncellulytic bacterium. A numberof additional biochemical and physiological tests were performed. DNA homology studies detected a 77.8%similarity to Cytophaga aquatilis (ATCC 29551). Comparisons of cellular fatty acid and carbohydrate contentsof isolate WF-164 with a Flexibacter sp., several Cytophaga spp., and Flavobacterium reference strains revealedsimilar patterns to that of C. aquatilis. On the basis of these characteristics, isolate WF-164 was identified as anew Cytophaga sp.

Workers at a textile facility experienced an outbreak of alung disease similar to hypersensitivity pneumonitis (HP).Several investigations were undertaken to identify thesource of the causative agent (25, 48, 65). Subsequentstudies demonstrated that an endotoxin-RNA complex re-covered from the biomass present in a water spray air-cooling system was the major serologically detectable anti-gen and a putative agent of the disease (17, 18).HP is a complex immunological reaction associated with

the inhalation of organic material from the environment.Antigenic agents have been reported to be produced bymany organisms, including bacteria (13, 55), fungi (5), andprotozoans (15). Three cases of HP in office workers result-ed from contact with the lipopolysaccharide (LPS) producedby several gram-negative bacteria that were tentativelyidentified as Flavobacterium spp. (54). A characteristic HPreaction in guinea pigs occurs after exposure to the LPSs ofspecies of Enterobacter, Klebsiella, and Pseudomonas, butnot of Agrobacterium or Xanthomonas (23), although theresults indicate that only a specific portion of the LPScomplex is responsible for the pathological responses in thedisease process.The objective of this study was to isolate representative

organisms from selected textile plant air-cooling units, pre-pare axenic cultures for antigenic screening, and identifythose which had been implicated as possible sources of thesuspect endotoxin. One bacterial isolate yielded a stronglypositive serological reaction to pooled sera which demon-strated the presence of precipitating antibodies from biopsy-documented HP patients (18). This isolate, designated WF-164, was identified by morphological and biochemical traits,as well as genetic similarity to reference strains.

MATERIALS AND METHODSSample collection. Representative samples of circulating,

chilled water (17°C, pH 6.4) and accumulated biofilm werecollected in sterile glass containers from selected locations inthe air-cooling units. All samples were immediately trans-

* Corresponding author.

936

ported to the laboratory and processed within 1 h of collec-tion. Air filters were also submitted for analysis.

Isolation of microorganisms. Water, slime, and homoge-nized air filter samples were diluted in phosphate-buffereddistilled water (0.3 mM KH2PO4, pH 7.4) and triplicatesamples of appropriate dilutions were spread onto selectivemedia for the cultivation of bacteria, fungi, and slime molds.The plating media employed for bacteria were plate count(PC) agar, nutrient agar (NA), MacConkey agar, eosinmethylene blue (EMB) agar, Endo agar (Difco Laboratories,Detroit, Mich.), chocolate agar, and blood agar (Biocon,Inc., Pensacola, Fla.). The plating media employed for fungiand slime molds were Sabouraud dextrose agar and oatmealagar (Difco), respectively. Culture plates of each mediumwere prepared in triplicate and incubated at 20°C for 14 days,35°C for 48 h, and 8°C for 21 days. After incubation, colonieswere selected, purified, and presented for antigenic screen-ing.

Antigenic screening. An indirect immunofluorescent anti-body assay (18), using pooled sera from biopsy-documentedHP patients, was employed to screen representative cul-tures. Serologically reactive isolates were cultured in half-strength nutrient broth (NB). LPS was extracted by thephenol-water method (70). All extracts were tested forserological reactivity by counter immunoelectrophoresis(18). Limulus amoebocyte lysate assays (18) were also usedto assess the biological endotoxin potential of the extract.

Reference strains. The reference strains listed in Table 1were obtained from the American Type Culture Collectionand Centers for Disease Control.

Morphological and cultural characteristics. Dimensions,motility, and arrangement of cells were observed by use oflight microscopy. Gliding cells were determined by the useof hanging-drop and wet mount preparations and by themethod described by Perry (47). Gram stains were per-formed on fresh cultures from NA plates.

Transmission electron microscopy (TEM) was used toobserve whole mounts of isolate WF-164 after 1, 5, and 10days of growth in half-strength NB and 1% peptone water(24) and on nutrient and PC agar. Negative staining of whole

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TABLE 1. Sources and DNA base ratios of reference strains

Organism Sourcea Strain G+C mol%no. (reference)

C. aquatilis Strohl and Tait ATCC 29551 33.7 (64)

C. johnsonae Stanier ATCC 17061 33.5 (37)

Flexibacter aurantiacus Lewin ATCC 23107 31.5 (38)

Flavobacterium spp.F. pectinovorum Dorey ATCC 19366 32.9 (42)F. breve (Lustig) Bergey et al. ATCC 14234 26 (69)F. meningosepticum King ATCC 13253 36.4 (42)F. multivorum Holmes CDC B5533 39.6 (28)F. capsulatum Leifson ATCC 14666 63 (42)a ATCC, American Type Culture Collection; CDC, Centers for Disease

Control.

mounts was performed with 2.0% aqueous ammonium mo-lybdate (12).

Detailed morphological an ultrastructural characteristicsof isolate WF-164 were observed by TEM. Cells werecultivated for 48 h in half-strength NB harvested by centrifu-gation, and washed once in 0.15 M cacodylate buffer (pH7.3). Cells were fixed with 1.2% (vol/vol, final concentration)glutaraldehyde, suspended in cacodylate buffer, and stainedwith 0.005% (wt/vol, final concentration) ruthenium red (35).After a 1-h fixation period, the cells were washed three timesfor 10 min each in cacodylate buffer enrobed in 4% (wt/vol,final concentration) gelatin and then cut into 1-cm3 blocks.The cells were postfixed in a solution of osmium tetroxideand ruthenium red (35). After successive 10-min washes in0.15 M cacodylate buffer, 0.075 M cacodylate buffer, anddistilled water, the gelatin blocks were dehydrated in a seriesof increasing concentrations of ethanol, thrice exposed topropylene oxide, and embedded in Epon (34). Thin sectionswere cut with glass knives on an LKB Ultrotome I andmounted on 300-mesh copper grids. After staining withuranyl acetate (63) for 10 min and lead citrate (53) for 3 min,thin sections were examined with a Philips EM201 transmis-sion electron microscope operated at 60 kV.Colony form and pigmentation were observed when the

organism was grown on Cook Cytophaga agar (10) and vy/2agar (21). Pigments produced by the isolate were extracted in100% acetone (64), and the absorption spectrum was deter-mined by using a Spectronic 20 spectrophotometer (Bauschand Lomb Instruments and Systems, Rochester, N.Y.). Todetect flexirubin-type pigments, colonies grown on Cy agar(21) were covered with 20% KOH; a change from yellow tored indicated the presence of flexirubin-type pigments (52).The ability of the isolate to form microcysts or fruitingbodies was evaluated by the methods of Dworkin and Voelz(14) or Carlson and Pacha (7), respectively.

Physiological characteristics. Temperature and pH optimawere determined by incubation of the organism in NB atselected temperatures and pHs and observation for visiblegrowth. Salinity tolerance was determined by inoculatingcultures of isolate WF-164 in NB amended with selectedconcentrations of NaCl and observing them for turbidity.Growth on EMB and MacConkey (Difco) agars were alsoevaluated.Anaerobic growth was tested in tubes of Penassay broth

(Difco antibiotic medium no. 3) containing 0.1% KNO3 inhalf-strength NB and on PC agar slants incubated in GasPakjars (BBL Microbiology Systems, Cockeysville, Md.) underhydrogen and carbon dioxide.

Biochemical characteristics. All biochemical tests wereperformed at room temperature (ca. 23°C). The methods ofLewin and Lounsbery (33) were employed for the determina-tion of alginate, cellulose, carboxymethyl cellulose, and agarhydrolysis. Chitinase activity on chitin agar medium (1) wasalso determined, allowing 14 days for incubation. NA plus0.2% soluble starch and NA plus 0.4% gelatin were devel-oped with Gram iodine solution and Frazier solution afterinoculation and a 48-h incubation to test for amylase andgelatin hydrolysis, respectively. NA plus 10% skim milk wasinoculated and, after a 48-h incubation, was observed forcaseinase activity. Positive results for the chitinase, amy-lase, gelatinase, and caseinase tests were indicated by a clearzone surrounding the area of growth. Carbohydrate metabo-lism was determined by the production of acid in media thatwas aerobically and anaerobically (sealed with sterile Vas-par) incubated for 2 weeks. Media employed were Board andHolding medium (2) amended with 0.5% selected carbohy-drates and Hugh-Leifson medium (Difco) amended with1.0% selected carbohydrates. An acid reaction was recordedas positive. Cytochrome oxidase was determined by testingfor oxidation of tetramethyl-P-phenylenediamine dihydro-chloride. The lead acetate strip test (59) was used to detectH2S, and phenylalanine agar (Difco) was used to detectphenylalanine deaminase activity. Nitrate reduction wasdetermined with Penassay broth containing 0.1% KNO3 (8)incubated aerobically and in GasPak jars under hydrogenand carbon dioxide. Daily spot checks up to 10 days wereperformed by using the qualitative method of detectingnitrate reduction as described in the Manual ofMethods forGeneral Bacteriology (59). DNase test agar (Difco) wasspotted with a 24-h culture for DNase activity. Clearingaround the area of growth after flooding the plate with 1 NHCI was recorded as positive. Lecithinase activity, deter-mined by using egg yolk agar (59), was demonstrated by aclearing around the colony after 4 days of growth.

Arginine dihydrolase, lysine decarboxylase, ornithine de-carboxylase, ,-galactosidase, and urease production weredetermined by API-20E strips (Analytab Products, Inc.,Plainview, N.Y.). All other tests were performed by usingstandard methods (59).

Antibiotic susceptibility. Antibiotic susceptibility patternswere determined by the Kirby-Bauer method according tothe instructions of the manufacturer of the Sensi-Disc micro-bial susceptibility test disks (BBL).

Lipid analysis. The fatty acid content of the isolate wascompared with those of eight reference strains (Table 1).Cells were harvested from a 48-h half-strength NB cultureand lyophilized. Cellular fatty acid profiles (16, 43, 44) wereprepared by saponification of 4.0 mg of dried bacterial cellswith 5% (wt/vol) NaOH and 50% aqueous methanol. Theliberated fatty acids were neutralized with 2.0 N HCl andextracted with chloroform-hexane (1:5, vol/vol). After dry-ing, the fatty acids were converted to the methyl ester with10% BF3-methanol at 70°C for 15 min. The methyl esterswere then removed by three extractions with chloroform-hexane (1:5, vol/vol) and concentrated to 100 ,ul by nitrogenevaporation at room temperature. Samples (5.0 ,ul) wereused for injection into a Varian model 2100 gas chromato-graph equipped with flame ionization detectors and dualdifferential electrometers. For lipid analysis, glass columns(12 ft [ca. 3.66m] by 2 mm [inside diameter]) packed with 3%SP-2100 DOH were used. After sample injection at 150°C,the instrument was programmed for 250°C (4°C/min), using asensitivity of 4 x 10-10 A full-scale. Nitrogen at a flow rateof 20 ml/min was employed as the carrier gas.

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FIG. 1. Negative stain of isolate WF-164 grown in nutrient broth for 24 h. Bar, 1 p.m; x13,500.

Carbohydrate analysis. The carbohydrate content of iso-late WF-164 was compared with those of five referencestrains. Carbohydrates from whole cells were analyzed astheir alditol acetate derivatives (45, 58) by modified gas-liquid chromatography (F. H. Deck and J. W. Cooper,manuscript in preparation). Identification of componentswas confirmed by comparison with authentic standards,using mass spectrophotometry.

Determination ofG+C moles percent. For determination ofthe content of guanine plus cytosine (G+C), DNA wasextracted from a 24-h culture of isolate WF-164 by a modi-fied Marmur procedure (31, 39) in which the first step was aphenol extraction. The midpoint of the hyperchrome shiftwas determined by employing a Gilford 2400-02 recordingspectrophotometer (Gilford Instrumentation Laboratories,Oberlin, Ohio) equipped with a Poly-Temp 80 circulatingwater bath (Polysciences, Inc., Warrington, Pa.). The absor-bance of the DNA solution (20 mg/ml in 0.15 M NaCl plus0.015 M sodium citrate buffer) (40) was measured at 260 nmin 1-cm quartz cuvettes. A 1°C/10 min rate of temperature

increase was employed and controlled manually. Resultswere corrected for thermal expansion (36).DNA homology studies. DNA samples were extracted from

isolate WF-164 and Cytophaga aquatilis (see above), sus-pended in 0.30 M NaCl plus 0.30 M sodium citrate buffer,and sheared by repeated passage through a 26-gauge hypo-dermic needle (56). The DNA solution (ca. 60 ,ug/ml) wasseated in a quartz cuvette and thermally denatured byheating to a temperature ca. 2°C above the temperature atwhich the absorbance increase at 260 nm ceased. Thespectrophotometer chamber was then rapidly cooled to atemperature 25°C lower than the melting temperature (Tm).The optical density of the DNA solution after the tempera-ture decrease was used as 0% renaturation. A 100% renatur-ation corresponded to the optical density of the DNAsolution at a Tm of 25°C before the hyperchrome shift. C0tvalues, i.e., the concentration ofDNA (moles of nucleotidesper liter of DNA solution) x time (seconds), were obtainedfrom a plot of log Cot versus percent reassociation (4). Thepercent homology was determined from the fractional C0t* 0 2 - 0 0 w 0 ff :$, :~~t.3 v0 ,,

*i ' W s > < i ' X w * _ | 0 X * g ~~~~v * ., , i Ai

FIG. 2. Thin section of isolate WF-164 fixed in the presence of ruthenium red and poststained with uranyl acetate and lead citrate. Arrowindicates inset. Bar, 0.5 ,um; x45,000. (Inset) Note the outer membrane (om), peptidoglycan layer (p), and the cell membrane (cm). Bar, 0.1,um; X100,000.

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TABLE 2. Biochemical characteristics of isolate WF-164

Characteristic Resulta

Polysaccharide degradationCellulose.Carboxymethyl cellulose.Chitin.+Agar.Alginate.Casein.+Starch.+Gelatin.+

Carbohydrate utilization'Glucoseoxidation.Lactose oxidation.Maltoseoxidation.. +Cellobiose oxidation .. +Sucrose oxidation.Arabinose oxidation .. +Glucose fermentation.

OtherCytochrome oxidaseproduction.+Catalase production.. +P-Galactosidase production.. +Phenylalanine deaminase.Arginine dihydrolase.Lysine decarboxylase.Ornithine decarboxylase.Urease production.DNase production.Hydrogen sulfide production.Lecithinase production.Acetylmethycarbinol production.Methyl redtest.Indole production.Nitrate reduction.

Antibiotic susceptibility (amt)cAmpicillin (10 p.g).Bacitracin (10 U).Carbenicillin (50 p.g) .+Cephalothin (30 ,gg).Chloramphenicol (30gig).+Gentamicin (10 VLg).Neomycin (30 gig).Penicillin (10U).Polymyxin B (300U).Streptomycin (10 g)...Tetracycline (30g)g).+a +, Positive result; -, negative result.b Production of acid in Board and Holding medium (2).+, inhibitory; -, not inhibitory to isolate WF-164.

values in the linear portion of the reassociation curve by theequation of Seidler and Mandel (57).

RESULTS

More than 700 bacterial isolates, in addition to the pre-dominant fungi and slime molds, were recovered from waterand slime samples from air-cooling units. Representativecultures were screened by the immunofluorescence tech-nique. The bacterial isolate, designated WF-164, was theonly isolate which contained a biologically active endotoxinand was both immunofluorescence and counter immunoelec-trophoresis positive.

Isolate WF-164 was determined to be a gram-negative,elongated, flexious bacillus. TEM-negative staining of wholemounts with 2.0% ammonium molybdate revealed nonflagel-

lated cells measuring 0.3 by 3.5 to 9.0 pum. After 24 h ofgrowth at room temperature (ca. 23°C) on NA, the cells hadan average length of 3.5 p.m; in NB, the average cell lengthmeasured 4.4 p.m (Fig. 1), although longer cells were ob-served after being grown in 1% peptone water. The averagecell length was 8.9 p.m after 24 h of growth.The ultrastructure of the organism revealed a bacillus

having a gram-negative cell envelope with a trilaminar innermembrane (cytoplasmic membrane), an intermediate layer(peptidoglycan), and a convoluted, trilaminar outer mem-brane (LPS). As previously observed (19, 46), we found anincreased density of the cell wall in TEM preparationstreated with ruthenium red. Vesicular tubular structuressimilar to those observed on the surfaces of Cytophagajohnsonae C4 (20) and Flexibacter sp. strain BH3 (29) wereobserved on the surfaces of isolate WF-164 (Fig. 1 and 2).

Gliding and flexing movements were observed by lightmicroscopy on solid surfaces and in liquid cultures. Typicalspreading, fingerlike projections of the bacterial colonieswere observed on both Cytophaga and vy/2 agars. Nofruiting bodies or microcysts were observed. In older cul-tures, autolysis and spheroplasts were observed. The orga-nism produced a bright yellow pigment when grown on allculture media. The extracted pigment demonstrated a maxi-mum absorption peak at 450 nm, suggesting either a carot-enoid or a flexirubin-type pigment. The KOH-flexirubin testwas positive.

Strain WF-164, originally isolated on PC agar at 20°C, alsogrew at 10 or 30°C but not at 4 or 37°C. Growth was observedwithin the pH range from 5.5 to 9.5 and in NB with 1.5%NaCl but not with 3.0% NaCl. Growth occurred on EMBagar but not on MacConkey agar. Anaerobic growth wasobserved in half-strength NB and on PC agar after 48 h.However, noticeably more luxuriant growth occurred on thesame media incubated aerobically.Of the macromolecules examined, chitin, starch, carboxy-

methyl cellulose, casein, and gelatin were hydrolyzed,whereas cellulose, agar, and alginate were not hydrolyzed.Results of the other biochemical tests are given in Table 2.Of the 11 antibiotics tested, chloramphenicol, carbenicillin,and tetracycline were inhibitory to isolate WF-164 (Table 2).Comparisons of the cellular fatty acid content of isolate

WF-164 with those of eight reference strains (Fig. 3) re-vealed a lipid profile similar to those of C. aquatilis, C.johnsonae, and also Flexibacter aurantiacus. Little differ-ence in the relative concentrations of straight- and branched-chain fatty acids of isolate WF-164 and the three referencestrains was detected. Methyltetradecenoic acid was themajor lipid (45% of the total fatty acids) detected in all fourof these strains. However, there were differences in therelative concentrations of straight- and branched-chain fattyacids of isolate WF-164 and the species of Flavobacterium.

Whole-cell carbohydrate composition demonstrated littlesimilarity between isolate WF-164 and any of the Flavobac-terium reference strains (Fig. 4). Isolate WF-164 contained ahigher percentage of N-acetyl-D-galactosamine (20.9%) thanthe reference Cytophaga spp. and Flavobacterium spp.Also, unlike the other reference strains analyzed, the highestpercentage of total carbohydrates of isolate WF-164 wascomposed of two components, heptose and dideoxyhexos-amine, which eluted at the same retention time on packedcolumns.

Finally, isolate WF-164 was determined to have a DNAbase composition of 34.8 mol% of G+C (mean of fivedeterminations). DNA homology studies demonstrated a77.8% similarity of isolate WF-164 to C. aquatilis.

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DISCUSSION

Many investigators are reluctant to use gliding motility asa major criterion to separate the Cytophaga and Flavobac-terium genera because of the ambiguity of the trait and theinconsistencies of the various methods used to determinegliding motility (9, 22, 27, 41, 42, 49, 60, 68). Cytophaga andFlavobacterium species are characterized by yellow pigmen-tation, overlapping G+C values (G+C content of DNA forFlavobacterium spp. ranges between 31 to 40 mol% (26), andsimilar morphological and biochemical traits (69). Becauseof these similarities and because yellow-pigmented bacteriatentatively identified as Flavobacterium spp. have beenimplicated in a previous outbreak of work-related lungdisease (54), lipid and carbohydrate analyses were per-formed to determine the taxonomic position of isolate WF-164.The cellular lipid profiles of isolate WF-164 and the

reference Cytophaga and Flexibacter strains, especially C.aquatilis, were similar. These lipid profiles were dominatedby nonhydroxylated, branched-chain compounds and were

comparable with the findings of other investigators who haveexamined Cytophaga spp. by using alkaline hydrolysis meth-ods (66, 67). Analyses of the lipid profiles of Flavobacteriumbreve and Flavobacterium meningosepticum, two specieswhich conform to the amended description of the genusFlavobacterium (26), were characterized by the presence ofa straight-chain fatty acid (12:0) that was absent in theCytophaga and Flexibacter reference strains as well as inisolate WF-164. Furthermore, the two species of Flavobac-terium lacked the branched-chain fatty acid, methyltetrade-cenoic acid, which was present in the Cytophaga andFlexibacter reference strains as well as in isolate WF-164.The carbohydrate analyses of the two Flavobacterium

reference strains were similar to each other but differed fromisolate WF-164 and the Cytophaga and Flexibacter strains inthat they lacked the heptose-dideoxyhexosamine fraction.Approximately 40% of the total cellular carbohydrate wasribose in the Flavobacterium strains, whereas the Cyto-phaga and Flexibacter strains contained less than 5% ribose.Both lipid and carbohydrate analyses indicated that isolateWF-164 was not a Flavobacterium sp.

In terms of morphology, isolate WF-164 conformed to thedescription of the class Flexibacteriae, the gliding bacteria(51, 62). Within this class, the absence of fruiting bodies anda low G+C value of 34.8 mol% eliminated isolate WF-164from the order Myxobacterales, which characteristicallyforms fruiting bodies and has a much higher range of G+Cvalues, i.e., 69 to 71 mol% (37, 51, 62). Cytophagales, theother order within this class, consists of six genera of whichonly two, Cytophaga and Flexibacter, were considered sincethe remaining four genera, Sphaerocytophaga, Microscilla,Sporocytophaga, and Lysobacter are described respectivelyas anaerobic, marine, possessing microcysts, and having ahigh G+C value, i.e., 65 mol% (11, 51).

Classically, the major criterion distinguishing Cytophagafrom Flexibacter species is the ability to degrade macromol-ecules (32, 61, 62). Furthermore, Flexibacter spp. havesomewhat higher G+C values (44 to 51 mol%) than doCytophaga spp. (28 to 39 mol%) (24, 51). Recent investiga-tions have also added the characteristic of cellular shapechange to distinguish Flexibacter from Cytophaga species(24, 51). Young, liquid cultures of Flexibacter spp. typicallyproduce long, threadlike cells that shorten with age. Sinceisolate WF-164 degraded chitin, carboxymethyl cellulose,gelatin, casein, and starch and since no extreme shape

NON-HYDROXYLATED FATTY ACIDS HYDROXY-

STRAIGHT BRANCHED P9 q Q0O q _ O Q O _ ~~~~~N le e °n In =OO

N le In LO 40 e *co co 9X X ?csc

WF-164

Cytophagaaquatilis

Cytophagajohnsonae

Flexibacteraurantiacus

Flavobacteriumpectinovorum

Flavobacteriumcapsulatum

Flavobacterium Imultivorum

Flavobacteriummeningosepticum

70

60-50-40-30

Flavobacterium 20breve _l0

FIG. 3. Fatty acid patterns of isolate WF-164 and referencestrains C. aquatilis (ATCC 29551), C. johnsonae (ATCC 17061), F.aurantiacus (ATCC 23107), Flavobacterium pectinovorum (ATCC19366), Flavobacterium capsulatum (ATCC 14666), Flavobacteriummultivorum (CDC B5533), Flavobacterium meningosepticum(ATCC 13253), and Flavobacterium breve (ATCC 14234).

change was observed, these data suggest that isolate WF-164is not likely to be a species of Flexibacter. The G+C ratio of34.8 mol% further supports the conclusion that isolate WF-164 is a Cytophaga species.Attempts to determine the species of isolate WF-164 by

species-specific properties as described in Bergey's Manual(69) were partially successful. Several species of Cytophagacould be eliminated from consideration because of the abilityto hydrolyze agar or cellulose or the inability to hydrolyzechitin. In most respects, isolate WF-164 exhibited character-istics similar to those described for C. johnsonae (8, 9, 61,62). The type strain, C. johnsonae ATCC 17061, degradesthe same macromolecules as did isolate WF-164, with theexception of alginate, yet differs in the production of hydro-gen sulfide, nitrate reduction, and fermentation of glucose(8). Unlike all nine strains of C. johnsonae studied by

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FIG. 4. Carbohydrate patterns of isolate WF-164 and referencestrains C. aquatilis (ATCC 29551), C. johnsonae (ATCC 17061), F.aurantiacus (ATCC 23107), Flavobacterium meningosepticum(ATCC 13253), and Flavobacterium breve (ATCC 14234).

Christensen (8), isolate WF-164 did not produce acid inlactose. Seven of these nine strains of C. johnsonae produceacid in sucrose, whereas isolate WF-164 did not.Our DNA homology studies suggested a very close rela-

tionship between isolate WF-164 and C. aquatilis. IsolateWF-164 was 77.8% homologous with C. aquatilis, a valuewhich would indicate enough similarity to place it in thesame genus, but not necessarily the same species, sinceclosely related strains within a genus are usually taken as

those that yield a relative binding ratio of 70% or greater (3,30). C. aquatilis and C. johnsonae mnay actually be the samespecies since two previously described C. aquatilis fishpathogens (64) are biochemically similar to C. johnsonae(UASME-1-25 and UASMB-1-25), which was described byChristensen (8). Although C. aquatilis (ATCC 29551) (64)and C. johnsonae (ATCC 17061) (8) degrade the same

macromolecules as did isolate WF-164, they differ in threeimportant biochemical characteristics, i.e., fermentation ofglucose, nitrate reduction, and hydrogen sulfide production.These differences suggest that although isolate WF-164 isclosely related to C. aquatilis and C. johnsonae, it is a

different species.Several other classification schema were employed to

describe isolate WF-164. By using the Hayes taxonomicstudy (22), we could place isolate WF-164 in "phenonseven." This cluster is represented by 41 strains, includingthe reference strains C. johnsonae (NCIB 10150), Cyto-phaga sp. (NCIB 9336), and Flavobacterium pectinovorum

(NCIB 9059). All of these strains exhibit gliding motility,grow as elongated rods (>8 Rxm) in NB, and have spreadingcolony types. G+C values of the members of this clusterrange from 31 to 38%.By using the Hirsch and Reichenbach (24) key characters

to classify Cytophaga-like bacteria, we found that isolateWF-164 resembled group A (a heterogeneous Cytophagagroup) with one exception: isolate WF-164 did not undergothe drastic cellular shape change characteristic of this group.

Strains of group D are reported to be similar to those ofgroup A, although they do not display cellular shapechanges. Organisms in group D, like isolate WF-164, pro-

duce catalase and cytochrome oxidase, are KOH positive,and have G+C values ranging from 32 to 36%. However,unlike group D, isolate WF-164 was lecithinase negative.Key characters described in The Prokaryotes (51) would

place isolate WF-164 in the family Cytophagaceae, genus

Cytophaga senu latiore (no. 6') a heterogeneous groupcomposed of several genera yet to be classified. Members ofthis group do not decompose cellulose, do not producemicrocysts or threadlike cells in young cultures, and are notobligate anaerobes. They have a G+C value which rangesbetween 30 and 35%, are isolated from freshwater or soil,and possess flexirubin-type pigments.The group of Cytophaga-like bacteria is certainly complex

as evidenced by the confusion among previous reports. G+Cand hybridization data (6, 24, 38, 50) indicate that there areprobably several different genera within this group. In viewof the unsettled nature of the genus, there is a certainreluctance to assign a specific name to isolate WF-164, as thelack of adequate comparative studies makes it difficult todistinguish between species. However, in light of the medi-cal importance of isolate WF-164 and since the preponder-ance of the data strongly supports the contention that isolateWF-164 is a unique, previously undescribed species withinthe genus Cytophaga, we propose the nanme Cytophagaallerginae. (The bacterium has been assigned the accessionnumber ATCC 35408.)

This is the first report of a Cytophaga species implicatedin an industrial health-related disease. Air- and water-cool-ing and humidifying systems have the potential to createhealth problems (such as those observed and well-docu-mented with Legionella species) by providing an environ-ment where human pathogens may develop. These aquaticenvironments need to be recognized as potential sources ofpublic and industrial health problems.

ACKNOWLEDGMENTSThis research was supported by a grant from Monsanto Fibers and

Intermediates, Inc.We thank P. A. Winter and Dan Doughtie for their valuable

assistance and advice in the electron microscopy studies and LesterBynum, W. B. Witmer, Larry Smith, and especially K. Bishop fortheir cooperation in sample collection and logistical support. Thanksare also extended to Dave Tison and John Riehm for their advicewith the DNA studies and to Darlene Roszak-MacDonell for per-formance of G+C moles percent determinations and DNA homolo-gy studies. Special thanks go to F. L. Singleton, who assisted incoordinating some of the work. We also thank Carney Hamilton,Joyce Boyce, and Luann Piazza for assistance in preparation of themanuscript.

LITERATURE CITED1. Bennett, C. B., and M. A. Hood. 1980. Effects of cultural

conditions on the production of chitinase by a strain of Bacillusmegaterium. Dev. Ind. Microbiol. 21:357-364.

2. Board, R. G., and A. J. Holding. 1960. The utilization of glucose

CARBOHYDRATE ANALYSES

E

It t B a- i { 6 | I S g g §

WF-164

Cytophaga

aquatilis _ - _

Cytophaga

jphnsonae

Flexibacter

aurantiacus_

Flavobacterium

meningosepticum50-40-

Flavobacterium 20-breve 10-

.~~~~~~~~~~~~~0

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by aerobic gram-negative bacteria. J. Appl. Bacteriol. 23:xi-xii.3. Brenner, D. J., G. R. Fanning, K. E. Johnson, R. V. Citarella,

and S. Falkow. 1969. Polynucleotide sequence relationshipsamong members of Enterobacteriaceae. J. Bacteriol. 98:637-650.

4. Britten, R. J., and E. Kohne. 1968. Repeated sequences inDNA. Science 161:529-540.

5. Buechner, H. A. 1966. Clinical aspects of fungus disease of thelungs including laboratory diagnosis and treatment, p. 123. InA. L. Banyai (ed.), Advances in cardiopulmonary diseases, vol.4. Yearbook Medical Publications, Chicago.

6. Callies, E., and W. Mannheim. 1980. Deoxyribonucleic acidrelatedness of some menaquinone-producing Flavobacteriumand Cytophaga strains. Antonie Leeuwenhoek J. Microbiol.46:41-49.

7. Carlson, R. V., and R. E. Pacha. 1968. Procedure for theisolation and enumeration of myxobacteria from aquatic habi-tats. Appi. Microbiol. 16:795-796.

8. Christensen, P. 1977. Synonymy of Flavobacterium pectino-vorum Dorey with Cytophaga johnsonae Stanier. Int. J. Syst.Bacteriol. 27:122-132.

9. Christensen, P. J. 1977. The history, biology, and taxonomy ofthe Cytophaga group. Can. J. Microbiol. 23:1599-1653.

10. Christensen, P. J., and F. D. Cook. 1972. The isolation andenumeration of cytophagas. Can. J. Microbiol. 18:1933-1940.

11. Christensen, P. J., and F. D. Cook. 1978. Lysobacter, a new

genus of nonfruiting, gliding bacteria with a high base ratio. Int.J. Syst. Bacteriol. 28:367-393.

12. Cole, R. M., and T. J. Popkin. 1981. Electron microscopy, p.

34-51. In P. Gerhardt (ed.), Manual of methods for generalbacteriology. American Society for Microbiology, Washington,D.C.

13. DeMaria, T. F., and R. Burnell. 1980. Effects of inhaledendotoxin containing bacteria. Environ. Res. 23:87-89.

14. Dworkin, M., and H. Voelz. 1962. The formation and germina-tion of microcysts in Myxococcus xanthus. J. Gen. Microbiol.28:81-85.

15. Edwards, J. H., A. J. Griffiths, and J. Mullisn. 1976. Protozoa as

sources of antigen in humidifier fever. Nature (London)264:438-439.

16. Fautz, E., G. Rosenfelder, and L. Grotjahn. 1979. Isobranched2- and 3-hydroxy fatty acids as characteristic lipid constituentsof some gliding bacteria. J. Bacteriol. 140:852-858.

17. Flaherty, D. K., F. H. Deck, J. Cooper, K. Bishop, P. A.Winzenburger, L. R. Smith, L. Bynum, and W. B. Witmer.1984. Bacterial endotoxin isolated from a water spray airhumidification system as a putative agent of occupation-relatedlung disease. Infect. Immun. 43:206-212.

18. Flaherty, D. K., F. H. Deck, M. A. Hood, C. A. Liebert, F. L.Singleton, P. A. Winzenburger, K. Bishop, L. R. Smith, L. M.Bynum, and W. B. Witmer. 1984. A Cytophaga species endo-toxin as a putative agent of occupation-related lung disease.Infect. Immun. 43:213-216.

19. Fletsher, M., and G. D. Floodgate. 1973. An electron-microscop-ic demonstration of an acidic polysaccharide involved in theadhesion of a marine bacterium to solid surfaces. J. Gen.Microbiol. 74:325-334.

20. Follett, E. A. C., and D. M. Webley. 1965. An electron micro-scope study of the cell surface of Cytophagajohnsonii and some

observations on related organisms. Antonie van LeeuwenhoekJ. Microbiol. Serol. 31:361-382.

21. Gude, H. 1980. Occurrence of cytophagas in sewage plants.App. Environ. Microbiol. 39:756-763.

22. Hayes, P. R. 1977. A taxonomic study of flavobacteria andrelated gram-negative yellow pigmented rods. J. Appl. Bacteri-ol. 43:345-367.

23. Helander, I., M. Salkinoga-Salonen, and R. Rylander. 1980.Chemical structure and inhalation toxicity of lipopolysaccha-rides from bacteria on cotton. Infect. Immun. 29:859-862.

24. Hirsch, I., and H. Reichenbach. 1981. The Cytophaga-likebacteria: a search for key characters, p. 145-152. In H. Reichen-bach and 0. B. Weeks (ed.), The Flavobacterium-Cytophaga

group. GBF monograph series no. 5. Verlag Chemie, Weinn-heim.

25. Hollick, G. E., H. W. Larsh, J. C. Hubbard, and N. K. Hall.1980. Aerobiology of industrial plant air system: fungi andrelated organisms. Zentralbl. Bakteriol. 8(Suppl.):89-95.

26. Holmes, B., and R. J. Owen. 1979. Proposal that Flavobacteri-um breve be substituted as the type species of the genus in placeof Flavobacterium aquatile and emended description of thegenus Flavobacterium: status of the named species of Flavobac-terium. Int. J. Syst. Bacteriol. 29:416-426.

27. Holmes, B., and R. J. Owen. 1981. Emendation of the genusFlavobacterium and the status of the genus, p. 17-26. In H.Reichenbach and 0. B. Weeks (ed.), The Flavobacterium-Cytophaga group. GBF monograph series no. 5. Verlag-Che-mie, Weinnheim.

28. Holmes, B., R. J. Owen, and R. E. Weaver. 1981. Flavobacteri-um multivorum, a new species isolated from human clinicalspecimens and previously known as group Ilk, biotype 2. Int. J.Syst. Bacteriol. 31:21-34.

29. Humphrey, B. A., M. R. Dickson, and K. C. Marshall. 1979.Physicochemical and in situ observations on the adhesion ofgliding bacteria to surfaces. Arch. Microbiol. 120:231-238.

30. Johnson, J. L. 1973. Use of nucleic acid homologies in thetaxonomy of anaerobic bacteria. Int. J. Syst. Bacteriol. 23:308-315.

31. Johnson, J. L. 1981. Genetic characterization, p. 450-472. In P.Gerhardt (ed.), Manual of methods for general bacteriology.American Society for Microbiology, Washington, D.C.

32. Leadbetter, E. R. 1974. Order II. Cytophagales nomen novum,p. 99. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey'smanual of determinative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

33. Lewin, R. A., and D. M. Lounsbery. 1969. Isolation, cultivationand characterization of Flexibacteria. J. Gen. Microbiol.58:145-170.

34. Luft, J. H. 1961. Iliprovements in epoxy resin embeddingmethods. J. Biophys. Biochem. Cytol. 9:409-414.

35. Luft, J. H. 1971. Ruthenium red and violet. I. Chemistry,purification, methods of use for electron microscopy and mech-anisms of action. Anat. Rec. 171:347-368.

36. Mandel, M., L. Igambi, J. Bergendahl, M. Dodson, Jr., and E.Scheltgen. 1970. Correction of melting temperature and cesiumchloride buoyant density of bacterial deoxyribonucleic acid. J.Bacteriol. 101:333-338.

37. Mandel, M., and E. R. Leadbetter. 1965. Deoxyribonucleic acidbase composition of myxobacteria. J. Bacteriol. 90:1795-1796.

38. Mandel, M., and R. A. Lewin. 1969. Deoxyribonucleic acid basecomposition of Flexibacter. J. Gen. Microbiol. 58:171-178.

39. Marmur, J. 1961. A procedure for the isolation of deoxyribonu-cleic acid from microorganisms. J. Mol. Biol. 3:208-218.

40. Marmur, J., and P. Doty. 1962. Determination of the basecomposition of deoxyribonucleic acid from its thermal denatur-ation temperature. J. Mol. Biol. 5:109-118.

41. McMeekin, T. A., and J. M. Shewan. 1978. A review oftaxonomic strategies for Flavobacterium and related genera. J.Appl. Bacteriol. 45:321-332.

42. Mitchell, T. G., M. S. Hendrie, and J. M. Shewan. 1969. Thetaxonomy differentiation and identification of Cytophaga spe-cies. J. Appl. Bacteriol. 32:40-50.

43. Moss, C. W. 1975. Gas-liquid chromatography as an analyticaltool in microbiology. Public Health Lab. 33:81-83.

44. Moss, C. W., M. A. Lambert, and W. H. Merwin. 1974.Comparison of rapid methods for analysis of bacterial fattyacids. Appl. Microbiol. 28:80-85.

45. Oades, J. M. 1967. Gas-liquid chromatography of alditol ace-tates: applications to analyses of sugar and complex hydroly-sates. J. Chromatogr. 28:246-252.

46. Pate, J. L., and E. J. Ordal. 1967. The fine structure ofChondrococcus columnaris. I. Structure and formation of meso-somes. J. Cell Biol. 35:1-13.

47. Perry, L. B. 1973. Gliding motility in some non-spreadingflexibacteria. J. Appl. Bacteriol. 36:227-232.

942 LIEBERT ET AL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 19

Jan

uary

202

2 by

175

.202

.54.

131.

CYTOPHAGA SP. IMPLICATED IN LUNG DISEASE 943

48. Reed, C. E., M. C. Swanson, M. Lopez, A. M. Ford, J. Major,and W. B. Witmer, and T. B. Valdes. 1983. Measurements ofIgG antibody and airborne antigen to control an industrialoutbreak of H.P. J. Occup. Med. 25:207-210.

49. Reichenbach, H. 1981. Taxonomy of the gliding bacteria. Annu.Rev. Microbiol. 35:339-364.

50. Reichenbach, H., H. Behrens, and I. Hirsch. 1981. The classifi-cation of the Cytophaga-like bacteria, p. 7-15. In H. Reichen-bach and 0. B. Weeks (ed.), The Flavobacterium-Cytophagagroup. GBF monograph series no. 5. Verlag Chemie, Weinn-heim.

51. Reichenbach, H., and M. Dworkin. 1981. Introduction of thegliding bacteria, p. 315-379. In M. P. Starr, H. Stolp, H. G.Triiper, A. Balows, and H. G. Schelgel (ed.), The prokaryotes.Springer-Verlag, New York.

52. Reichenbach, H., H. Kleinig, and H. Achenbach. 1974. Thepigments of Flexibacter elegans: novel and chemosystematical-ly useful compounds. Arch. Microbiol. 101:131-144.

53. Reynolds, E. S. 1963. The use of lead citrate at high pH as anelectron opaque stain in electron microscopy. J. Cell Biol.17:208-212.

54. Rylander, R., P. Haglind, M. Lundholm, I. Mattsby, and K.Stenquist. 1978. Humidifier fever and endotoxin exposure. Clin.Allergy 8:511-516.

55. Rylander, R., and M. C. Snella. 1976. Acute inhalation toxicityof cotton plant dusts. Br. J. Ind. Med. 33:175-180.

56. Schleif, R. F., and P. C. Wensink. 1981. Practical methods inmolecular biology. Springer-Verlag, New York.

57. Seidler, R. J., and M. Mandel. 1971. Quantitative aspects ofdeoxyribonucleic acid renaturation: base composition, state ofchromosome replication, and polynucleotide homologies. J.Bacteriol. 106:608-614.

58. Shaw, D. H., and G. W. Moss. 1967. Quantitative estimation ofneutral sugars by gas-liquid chromatography. J. Chromatogr.41:350-357.

59. Smibert, R. M., and N. R. Krieg. 1981. General characteriza-tion, p. 409-443. In P. Gerhardt (ed.), Manual of methods forgeneral bacteriology. American Society for Microbiology,Washington, D.C.

60. Soriano, E., and R. A. Lewin. 1965. Gliding microbes: sometaxonomic reconsiderations. Antonie Leeuwenhoek J. Micro-biol. 31:66-79.

61. Stanier, R. Y. 1942. The Cytophaga group: a contribution to thebiology of myxobacteria. Bacteriol. Rev. 6:143-196.

62. Stanier, R. Y. 1957. Order VII. Myxobacterales Jahn, p. 854-891. In R. S. Breed, E. G. D. Murray, and N. R. Smith (ed.),Bergey's manual of determinative bacteriology, 7th ed. TheWilliams & Wilkins Co., Baltimore.

63. Stempak, J. G., and R. T. Ward. 1964. An improved stainingmethod for electron microscopy. J. Cell Biol. 22:697-701.

64. Strohl, W. R., and L. R. Tait. 1978. Cytophaga aquatilis sp.nov., a facultative anaerobe isolated from the gills offreshwaterfish. Int. J. Syst. Bacteriol. 28:293-303.

65. Swanson, M. C., C. E. Reed, and A. M. Ford. 1980. Applicationof a solid phase radioimmunological assay for IgG antibody inan industrial outbreak of H.P. due to contaminated chilled waterair conditioning system. J. Allergy Clin. Immunol. 65:228.

66. Walker, R. W. 1969. cis-11-Hexadecenoic acid from Cytophagahutchinsonii lipids. Lipids 4:15-18.

67. Ware, J. C., and M. Dworkin. 1973. Fatty acids of Myxococcusxanthus. J. Bacteriol. 115:253-261.

68. Weeks, 0. B. 1969. Problems concerning the relationships ofcytophagas and flavobacteria. J. Appl. Bacteriol. 32:13-18.

69. Weeks, 0. B. 1974. Genus Flavobacterium Bergey et al., p. 357-364. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey'smanual of determinative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

70. Westphal, O., and K. Jann. 1963. Bacterial lipopolysaccharides:extraction with phenol-water and farther applications of theprocedure. Methods Carbohydr. Chem. 5:83-92.

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